The PI and her postdoctoral fellows are acquiring real time dynamics data from a sensitive laser scanning microscope system with a built-in long-term tissue incubation capability. Currently we are characterizing RelA, RelB, and c-Rel dynamics in two different cell types with a panel of Toll-like receptor ligands and quantifying the time lapse data. We are also developing new knock-in mouse strains which express fluorescent Rel (NF-kappaB subunits) fusion proteins from the native genomic loci, by CRISPR-Cas genome engineering. Currently we are screening pups for correct expression and integration of the Rela targeting constructs. NF-kappaB dynamics will be monitored alone or in parallel with cytokines or lineage TFs by crossbreeding the Rel knock-in mice with existing mice such as fluorescent reporters of cytokines or transcription factors associated with distinct phenotypes. Many of these factors are co-activated during immune cell differentiation. Moreover, simultaneous live cell imaging of two transcription factors each with its own complex dynamics will reveal unprecedented insight about how separate dynamic networks interact with each other to achieve co-regulation of gene expression programs in immunity and cell differentiation.